International Journal of Molecular Sciences Review Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens Sabina K˛edzierska-Mieszkowska 1,* and Michal Zolkiewski 2 1 Department of General and Medical Biochemistry, Faculty of Biology, University of Gda´nsk, 80-308 Gda´nsk,Poland 2 Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA; [email protected] * Correspondence: [email protected]; Tel./Fax: +48-58-5236064 Abstract: This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB’s protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections. Keywords: bacteria; ClpB; human; infection; molecular chaperone; pathogen; virulence Citation: K˛edzierska-Mieszkowska, S.; Zolkiewski, M. Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens. Int. 1. Introduction J. Mol. Sci. 2021, 22, 5319. https:// Bacteria are one of the epidemiological agents that can cause infectious diseases in doi.org/10.3390/ijms22105319 humans. It is estimated that approximately 10% of the known infectious agents are bacte- ria [1]. However, it should be emphasized that the vast majority of bacteria are not harmful Academic Editors: Antonella to humans and some of them are beneficial, such as intestinal bacteria which facilitate Marino Gammazza and Celeste food digestion in the human gastrointestinal tract and are involved in the production of Caruso Bavisotto vitamins necessary for human physiology. Besides, bacteria play an important role in maintaining the ecological balance in the environment they occupy. Thus, only a fraction Received: 28 April 2021 of bacteria is pathogenic, and causes diseases in humans and animals. A disease mani- Accepted: 15 May 2021 Published: 18 May 2021 festation is a consequence of the bacteria prevailing over the defenses of a mammalian immune system. Diseases caused by pathogenic bacteria are as diverse as the bacteria themselves and include pneumonia, food- and water-borne infections, wound and blood- Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in stream infections (known as septicemia or sepsis), or sexually transmitted diseases, such as published maps and institutional affil- gonorrhea and chlamydiosis. Bacterial infectious diseases have a very significant impact iations. on public health [2]. Notably, lower respiratory infections, infectious diarrhea, and tubercu- losis, caused by bacterial pathogens, are among the top causes of human mortality in the world [3]. Some bacteria are responsible for zoonotic infections which can be transmitted from animals to humans via direct or indirect contacts, sometimes also by an invertebrate vector. Importantly, bacterial zoonoses cause millions of human deaths every year, which Copyright: © 2021 by the authors. has a significant impact on global public health and economies worldwide [4]. Bacterial Licensee MDPI, Basel, Switzerland. This article is an open access article zoonoses also cause major losses in livestock and impact the farming industry [5]. Farm an- distributed under the terms and imals are often reservoirs of zoonotic pathogens [4,6]. Therefore, a “One Health” approach conditions of the Creative Commons to both medical and veterinary care, promoted by the U.S. Centers for Disease Control and Attribution (CC BY) license (https:// Prevention, is essential for controlling the spread of zoonotic bacterial diseases. creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 5319. https://doi.org/10.3390/ijms22105319 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5319 2 of 12 Understanding how bacterial infections spread and how pathogenic bacteria outsmart the host’s defenses is crucial to controlling all bacterial diseases. During the infection process, an intense battle for survival takes place between an invading pathogen and the human body. The outcome of this battle depends on responses from both the pathogen and its host. In response to a bacterial intruder, a human host activates its defense and protective mechanisms, including the innate and the adaptive immune systems, which play a critical role in protection against a broad variety of pathogens, including bacteria. These two systems are sequentially activated during infection and work together to fight the invaders and destroy the initial infection [7]. In turn, pathogenic bacteria have evolved diverse strategies of interacting with the human host and rapid adaptation to new environmental conditions, including elevated temperature or oxidative stress, which are associated with the host’s immune responses. Thanks to these strategies, a bacterial intruder can establish itself within an infected host, i.e., it can survive, replicate and spread inside the human body, leading to a disease manifestation. Pathogenic bacteria use a number of virulence mechanisms and factors as effective weapons in subverting the human host and turning the host’s cellular processes to their advantage. The molecular chaperone ClpB which belongs to the Hsp100/Clp subfamily of the AAA+ ATPases (ATPases associated with diverse cellular activities) is one of the factors which can enhance bacterial survival in the host during infection. Hsp100 chaperones are present in bacteria, protozoa, fungi, and plants, but not in animals and humans. The aim of this review is to discuss the current understanding of the role of ClpB in selected pathogenic bacteria and its importance during bacterial infections in humans. Importantly, since ClpB is not found in human cells, this chaperone becomes a promising target for novel antibacterial strategies, which is particularly important nowadays due to an increasing rate of developing antibiotic resistance among pathogenic bacteria, an alarming problem for global public health [8,9]. 2. AAA+ Chaperone ClpB: Its Structure, Function and Mechanism of Action Bacterial ClpB and its yeast homolog, Hsp104 are the best-studied members of the Hsp100 chaperone family [10,11]. ClpB is found in the vast majority of bacteria with the ex- ception of distinct bacterial species, such as the Gram positive Bacillus subtilis [12,13]. This year marks three decades since ClpB was first identified as a heat shock protein necessary for the optimal growth and survival of E. coli at high temperature and thus required for the thermotolerance of this bacterium [14]. Unlike many other Clp family members that form complexes with peptidase subunits and participate in protein degradation, ClpB co- operates with the DnaK chaperone system to solubilize and reactivate aggregated proteins accumulating in bacteria under stress conditions [15–18]. ClpB can remodel some protein substrates without the DnaK assistance, but cooperation of ClpB and DnaK produces the most efficient disaggregation [19–21]. Interestingly, the ClpB-DnaK cooperation in protein disaggregation is species-specific, i.e., ClpB efficiently cooperates only with DnaK from the same microorganism [22–24]. Like other Hsp100 family members, ClpB forms ring-shaped hexamers in the pres- ence of ATP [25–28] with a narrow central channel (pore), wide enough to accommodate extended unfolded polypeptides (Figure1). Each protomer of ClpB consists of multiple domains: N-terminal domain (NTD), two ATP-binding modules, i.e., nucleotide-binding domain 1 (NBD-1) and nucleotide-binding domain 2 (NBD-2) and a unique coiled-coil middle domain (MD) inserted at the end of NBD-1 (Figure1A). Each NBD domain contains all the characteristic and highly conserved motifs of AAA+ ATPases, namely Walker A, Walker B, arginine finger, sensor-1, and sensor-2 (Figure1A). The NTD of ClpB is responsi- ble for the recognition and binding of protein substrates. NBDs generate energy from ATP for polypeptide translocation through the ClpB channel. The MD mediates the interactions with DnaK that are required for bacterial thermotolerance and efficient protein disaggrega- tion [23,24,29] and is also involved in coordinating the communication between NBDs [19]. Of note, the presence of the coiled-coil MD distinguishes ClpB from such Hsp100/Clp Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12 Int. J. Mol. Sci. 2021, 22, 5319 3 of 12 such Hsp100/Clp proteins as ClpA, ClpX or HslU that are associated with a peptidase proteins as ClpA, ClpX or HslU that are associated with a peptidase (ClpP or HslV) and do (ClpP or HslV) and do not display disaggregase activity. not display disaggregase activity. FigureFigure 1. 1. (A(A) )Structural Structural organization organization of of the the ClpB ClpB monomer. monomer. Four Four domains domains are are indicated: indicated: N N-terminal-terminal domaindomain (NTD), (NTD), two two nucleotide nucleotide-binding-binding domains domains (NBD (NBD-1-1 and and NBD NBD-2),-2), and and middle